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VETERINARY SERVICES MEMORANDUM NO. 800.104 

TO: Veterinary Services Leadership Team

 Directors, Center for Veterinary Biologics

 Biologics Licensees, Permittees, and Applicants

 

FROM: Jack A. Shere

 Deputy Administrator

SUBJECT: In Vitro Serial Release Potency Test for Completed Product Containing 

Clostridium chauvoei

I. PURPOSE

This memorandum provides guidance to licensees, permittees, and applicants concerning the 

Center for Veterinary Biologics (CVB) policy for obtaining an exemption to use an in vitro 

potency test in place of the current Standard Requirement (SR) test for releasing serials of 

product containing Clostridium chauvoei antigen. 

II. REPLACEMENT

This memorandum cancels Veterinary Services (VS) Memorandum No. 800.104, dated

May 29, 2003.

III. BACKGROUND

Currently, CVB requires testing of completed product containing Clostridium chauvoei 

antigen prior to serial release using a guinea pig vaccination-challenge potency test as 

specified in title 9, Code of Federal Regulations (9 CFR), part 113.106. The CVB encourages 

implementation of in vitro testing to replace the guinea pig vaccination-challenge potency 

test as part of our ongoing commitment to refine, reduce, and replace animal testing. 

The CVB-Policy, Evaluation, and Licensing (CVB-PEL) has developed an in vitro test for 

quantifying C. chauvoei antigen in completed product, which may be used as a serial release 

potency test for products containing inactivated C. chauvoei. This test is an antibody 

sandwich enzyme-linked immunosorbent assay (ELISA) that uses C. chauvoei flagellaspecific monoclonal antibody and C. chauvoei flagella-specific polyclonal rabbit antibodies 

to capture and detect protective flagellar antigen. 

The amount of flagellar antigen in the test serial is compared to that in an approved reference 

preparation using the in vitro test developed by CVB. The protocol, designated BBPRO0220

and entitled “Potency Testing of Clostridium chauvoei Bacterins using an ELISA Procedure,” 

is available onCVB web site and may also be requested directly from CVB-PEL. 

United States 

Department of 

Agriculture

Animal and Plant 

Health 

Inspection 

Service

Veterinary 

Services

Washington, DC 

20250

for
VETERINARY SERVICES MEMORANDUM NO. 800.104

Page 2

IV. POLICY

A. Exemption Request

According to 9 CFR 113.4, the licensee or permittee must comply with the test 

methods and procedures contained in all applicable SRs, unless the CVB-PEL has 

provided an exemption, and must cite any exemption in the filed Outline of 

Production for the product. The licensee or permittee submits a letter to CVB-PEL 

requesting an exemption from 9 CFR 113.106 SRs for Clostridium chauvoei

Bacterin. CVB-PEL will consider granting an exemption request if the firm has 

performed all of the following:

1. Test validation data

a. Products. The ELISA is validated for testing the specific products for which 

the test is intended and in accordance with VS Memorandum No. 800.112.

b. Correlation data. The ELISA correctly identifies potent and subpotent serials

when compared to the SR guinea pig vaccination-challenge test. The firm 

may submit data generated by testing diluted products used for reference

qualification as part of the test correlation data.

2. Changes to the Outline of Production

a. Test method. The BBPRO0220 procedure or an acceptable alternative ELISA

procedure for quantifying the flagellar antigen of C. chauvoei is described in 

section V.C. of the Outline of Production or a Special Outline.

b. Information regarding reference preparations. The identity, stability 

monitoring protocol, and expiration date of the approved reference

preparation is included in section V.C. of the Outline of Production.

c. Test criteria. The requirements for a satisfactory test, along with validity

requirements, are included in section V.C. of the Outline of Production. If

specified in section V.C. of the Outline of Production, the CVB-PEL allows 

the firm to use the guinea pig vaccination-challenge test described in the SR

as a second-stage test on serials failing the ELISA potency test. The firm must 

designate the ELISA test results as “Inconclusive” if the guinea pig 

vaccination-challenge test is used for second-stage testing.

d. Date of exemption. The exemption to the SR test is documented in section

V.C. of the Outline of Production in accordance with 9 CFR 113.4 and VS

Memorandum No. 800.206.
VETERINARY SERVICES MEMORANDUM NO. 800.104

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3. Qualified reference data

The firm has described the methodology for qualifying a reference. Section IV.B 

of this document describes the methodology required.

B. Qualification of C. chauvoei Reference for in vitro Potency Assay

1. Initial reference qualification. The initial reference qualification is applicable to 

products for which the in vitro potency assay has not previously been approved for 

serial release. The firm must make qualifying serials for all proposed reference

preparations according to a filed Outline of Production. The firm must prove the 

efficacy of references by a vaccination-challenge study that includes at least 10

vaccinated and 5 control cattle. All cattle are challenged with a virulent C.

chauvoei spore suspension. If at least 8 of 10 of the vaccinated cattle survive the

challenge, and at least 4 of 5 of the control cattle die as a result of the challenge,

the reference is satisfactory. CVB-PEL highly recommends evaluation in guinea 

pigs according to 9 CFR 113.106, as described below, concurrently with the 

initial reference qualification study in cattle to establish a guinea pig 50% 

protective dose (PD50).

The firm establishes the PD50 of the reference preparation in guinea pigs using the

vaccination-challenge methods outlined in Supplemental Assay Method 200 for

Potency Testing Products Containing Clostridium chauvoei Antigen. The firm 

vaccinates groups of at least 5 guinea pigs with fractional doses of the reference

preparation, using serial dilutions no greater than two-fold. CVB-PEL prefers the 

firm to establish the PD50 determination using the geometric mean of at least 5

independent assay replicates. CVB-PEL allows the firm to use the established 

guinea pig PD50 as a standard to requalify and/or monitor existing approved 

reference preparations and to qualify new reference preparations.

2. Reference qualification or requalification of previously approved reference. If a 

product has an established guinea pig PD50, CVB-PEL allows the firm to use the 

guinea pig vaccination-challenge for future reference qualification and 

requalification of an approved reference. If the firm has not established a guinea 

pig PD50, CVB-PEL requires the firm to perform future reference qualification 

and requalification of an approved reference in cattle.

C. Confirmatory Testing

CVB-PEL may conduct confirmatory testing on serials according to 9 CFR 113.106

after approval of the in vitro potency assay.
VETERINARY SERVICES MEMORANDUM NO. 800.104

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V. IMPLEMENTATION/ APPLICABILITY

Firms wishing to use the ELISA test for serial release should request an exemption through 

their assigned reviewer. The request should be accompanied by the data and documents 

described above.